The nature of polymorphism and allelism were re-evaluated in the minor lymphocyte stimulating (Mls) system. Mls determinants are defined by the ability to stimulate primary proliferative T cell responses between major histocompatibility complex (MHC) identical cells. Originally, Mls was described as a single locus system in- volving at least four polymorphic alleles. Proliferating T cell clones were generated which were specific for Mlsa, Mlsc, or Mlsd. These, in combination with primary proliferative T cell responses, were then employed to analyze the relationship between Mlsa, Mlsc, and Mlsd determinants. It was found that Mlsd cells appear to express the sum of Mlsa and Mlsc determinants. In addition, formal genetic analyses were carried out to identify the relationship between the genes encoding Mlsa, Mlsc, and Mlsd. It was found that Mlsa and Mlsc are encoded by non-allelic and in fact unlinked genes. Moreover, an Mlsd strain expresses independently the products of unlinked Mlsa-like and Mlsc-like genes. Thus, in contrast to previous understanding, the Mls system is composed of the products of at least two unlinked loci, with no evidence for structural polymorphism at either locus at the present time. An analysis of T cell receptor (TCR) expression in Mlsc reactive T cells demonstrated a striking association of this reactivity with expression of the V-beta3 gene product. This was true both for T cell clones generated by repeated stimulation wit Mlsc stimulators as well as for antigen specific T cells with coincidental reactivity to Mlsc. In addition, it was demonstrated that mouse strains which express Mlsc delete expression of V-beta3 in their mature peripheral T cell populations. This failure to express V- beta3 presumably reflects a consequence of the maintenance of tolerance to self Mlsc products. These findings provide a basis for the understanding of high T cell precursor frequency for Mlsc products, reflecting the overall expression of V-beta3 in the T cell repertoire. Moreover, they demonstrate the importance of these products in selection of the antigen-specific T cell repertoire.